The major histocompatibility complex (MHC) is a gene family whose function is to aid recognition of foreign antigens by the immune system. The mutation rate of the Kb gene of the mouse MHC was determined by skin grafting technique and was found to be very high while the mutation rate of the Kd gene was low. Kb mutants carry multiple rather than single nucleotide substitutions and are thought to be products of gene conversion events. However, skin grafting does not detect small alterations in Kb antigen caused by some base pair substitutions in the gene. Since parental mice of Kb mutants have not been tested by techniques other than skin grafting, they may have carried intermediate step mutations undetectable by skin grafting. Therefore the number of distinct genetic steps and the actual mechanism involved in Kb mutations remains obscure. We propose to determine how many genetic events have been involved in Kb mutants appearing de novo by molecular tests of the mutants and of their ancestors. The nature of the alterations in mutants and their progeny will be determined by Southern blots, Northern analysis and RNA sequencing technique, and after that we will test nucleic acids isolated from parental mice for the presence of the specific alteration found in the mutant. In the same experiment, we seek to determine the genetic region within the H-2 complex that controls high mutation rate of the Kb gene versus low rate of the Kd gene. We also want to determine sequence alterations in some unusual H-2a mutants silent in skin grafting tests and/or carrying concomitant class I plus class II MHC antigenic changes. This knowledge is important for fuller understanding of the mechanisms of gene diversification and evolution of MHC and for further definition of structure-function relationships in MHC molecules.